, Danvers, MA), and mouse anti-HepPar1 (hepatocyte paraffin-1 antigen) (1:100; Abcam). Appropriate secondary antibodies, DyLight 488 donkey anti-sheep immunoglobulin

G (IgG), DyLight http://www.selleckchem.com/products/BEZ235.html 549 donkey antirabbit IgG, and DyLight 594 donkey anti-mouse IgG (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used. In the control experiments, cells were stained with secondary antibodies only. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; Roche). Quantification of immunofluorescence (IF) staining was also performed for a few select markers. For this purpose, National Institutes of Health Image J software was used to measure the mean S1P Receptor inhibitor gray value (MGV) for staining of cells through the culture period. For each cell, the MGV of the area of interest (Sall4 and HNF4a: nuclei; HepPar1: whole cell) was calculated and background was subtracted. An average of the MGV

was then calculated for each group. Values were then extracted from that of negative control groups, which were only stained with secondary antibodies. Mouse liver tissues were fixed in 2% paraformaldehyde for 30 minutes, then embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA) compound.

Sections were sliced at a thickness of six microns. After air-drying, sections were incubated in β-galactosidase reporter gene staining solution (β-Galactosidase Reporter Gene Staining Kit; Sigma-Aldrich) at 37°C overnight, then counterstained with nuclear fast-red staining (Sigma-Aldrich) for 5 minutes at room temperature. For IF staining, sections were fixed MCE in cold acetone. Rabbit anti-β-galactosidase (1:20; CEDARLANE Labs, Burlington, NC) was mixed with either sheep anti-albumin antibody (1:500) or mouse anti-CK7 (1:100). After washing, DyLight 549 anti-mouse, DyLight 549 anti-sheep, or DyLight 488 anti-rabbit antibodies, at a dilution of 1:200, were used as secondary antibodies. Total RNA was isolated using a miniRNA kit (QIAGEN, Valencia, CA), according to instructions of the supplier, and was subsequently subjected to reverse-transcription polymerase chain reaction (RT-PCR). First, 1 μg of total RNA was reverse-transcribed in a 20-μL volume with the Superscript III Reverse Transcription Kit (Invitrogen, Carlsbad, CA), as per the manufacturer’s recommendations.